Abstract: The glucocorticoid-type steroids are the mainstay of asthma therapy. A subgroup of asthmatic patients develops steroid resistance, which becomes a major therapeutic challenge and imposes a high cost to the society. An understanding the mechanism of steroid resistance is important. Asthma is driven by the type 2 immune response. Type 2 innate lymphoid cells (ILC2s) are an important source of type 2 cytokines. Two epithelial cytokines?IL33 and TSLP promote the development and function of ILC2s. We have recently reported that airway ILC2s, as compared to blood ILC2s, from a subgroup of asthmatic patients are steroid resistant in a TSLP (thymic stromal lymphopoietin)-dependent manner. Blood ILC2s become steroid resistant when exposed to TSLP but not IL33. We have uncovered a mechanism for this dichotomous effect. Steroids upregulate (protagonize) the receptors for TSLP but not IL33. By upregulating the receptors steroids lower their activation threshold. Steroids still antagonize many inflammatory pathways in these cells but, surprisingly, protagonize the pathways that directly interfere with the formation of the glucocorticoid receptor repressor complex. Consequently, these pathways upregulate type 2 cytokines unabated in the presence of steroids, which results in steroid resistance. Based upon these findings we hypothesize that activation of steroid protagonized receptors induces steroid resistance through upregulation of select signaling pathways that disrupt the formation of the glucocorticoid receptor (GR) repressor complex. We propose 3 specific aims to test the foregoing hypothesis. Under specific aim 1 we will study the molecular mechanism of TSLP-induced steroid resistance of ILC2s. We will examine how a novel signaling pathway involving MEK2-CBX7-PRC1, identified in preliminary experiments, hinders the organization of the GR repressor complex. We will use human blood and lung ILC2s to maintain human disease relevance. Specific aim 2 is devoted to mechanistic and preclinical studies in mice. We will examine how repetitive exposure to allergens and rhinovirus contributes to the development of steroid resistance. We will use genetically modified mice to validate the importance of steroid resistant pathways. We will perform preclinical trials with pathway inhibitors in a humanized mouse model of steroid resistant asthma. Under specific aim 3 we will establish the relevance of TSLP and TSLPR signaling molecules for steroid resistance by studying bronchoalveolar lavage ILCs and T cells from steroid resistant and steroid sensitive asthmatic patients. We will evaluate pathway inhibitors for reversal of steroid resistance of airway lymphoid cells. We have all the necessary expertise, resources and collaborators to accomplish this project. The proposal is important because it addresses an unresolved clinical problem that affects not only asthma but also many chronic inflammatory, autoimmune and neoplastic diseases where steroid resistance is a therapeutic challenge. A successful completion of this project will help develop novel therapeutic modalities targeting steroid resistance, in general.